The present invention relates to an enzymatic process for the preparation of 7xcex2-(4-carboxybutanamide) cephalosporanic acid. More particularly, it describes a method for isolating the gene which codes for an enzyme with D-aminoacid oxidase activity by the use of recombinant DNA techniques, the cloning of said gene in a microorganism of the genus Escherichia, the modification of said enzyme by protein engineering techniques, the hyperproduction of said modified enzyme by fermentation in said microorganism and the extraction of the modified enzyme for preparation of 7xcex2-(4-carboxybutanamide) cephalosporanic acid. This acid is an intermediate compound for the preparation of 7-amino cephalosporanic acid, which in turn is a known intermediate for the preparation of a wide variety of antibacterial agents in the cephalosporins family.
For the production of 7xcex2-(4-carboxybutanamide) cephalosporanic acid, also called glutaryl-7-aminocephalosporanic acid (hereinafter referred to as GL-7ACA), from cephalosporin C, the use of the enzyme D-aminoacid oxidase (hereinafter referred to as DAO) from various microorganisms such as Trigonopsis variabilis (Biochem. Biophys. Res. Commun. (1993) 31: 709), Rhodotorula gracilis (J. Biol. Chem. (1994) 269: 179) and Fusarium solani (J. Biochem. (1990) 108: 1063) is known. The production of DAO by the use of these microorganisms involves many disadvantages. For one thing, the level of production of DAO activity is very low, and, for another, other undesirable enzyme activities such as esterases and catalases are present together with said enzyme. The former break down GL-7ACA acid, reducing the yield and thus increasing the costs of the purification process. The latter destroy the hydrogen peroxide needed in the catalysis and necessitate the addition of this compound, which also increases the costs of the process and at the same time causes a loss in the activity of the enzyme, reducing its possibilities of re-use. In order to avoid said enzymatic contamination it is necessary to purify the DAO activity, which greatly increases the costs and difficulty of the enzymatic process for obtaining GL-7ACA from cephalosporin C.
A process has recently been described for isolating the gene which codes for DAO in T. variabilis and expressing it in E. coli and in T. variabilis (Japanese Patent Application Laid-Open No. 71180/1988; European Patent Application No. 93202219.7, Publication No. 0583817A2). Moreover, the gene which codes for the DAO activity of F. solani has also been cloned and expressed in E. coli and Achremonium chrysogenum (Japanese Patent Application Laid-Open No. 2000181/1990; J. Biochem. (1990) 108: 1063; Bio/Technology (1991) 9: 188) and more recently the gene which codes for DAO in R. gracilis has been cloned and expressed in E. coli (Spanish Patent P9600906).
In view of the great interest that the availability of a purified DAO activity has industrially for the production of GL-7ACA, the objective of the present invention was centred on the production of an enzyme with DAO activity which could easily be purified. In this sense it is known that one of the most effective ways of purifying a protein is the use of affinity chromatography (Sassenfeld, H. M. (1990) Trends Biotechnol. 8: 88). For this purpose it is necessary to find a chromatographic support which allows the selective binding and/or elution of the protein of interest. Said chromatographic support must contain a recognition molecule or ligand for said protein in such a way that the interaction between it and the ligand is specific and strong enough to allow the selective elution of the protein. The development of an affinity chromatography support is not simple, and until now no process allowing the affinity purification of DAO enzyme activities in a single step has been described.
Furthermore it is known that certain genetic engineering processes allow the modification of proteins, improving particular properties which facilitate the purification thereof. Thus, various systems have been developed for modification of the structure of a protein to allow its affinity purification (Sii, D. and Sadana, A. (1991) J. Biotechnol. 19: 83; Narayanan, S. R. and Crane, L. J. (1990) Trends Biotechnol. 8: 12; Scouten, W. H. (1991) Curr. Opinion Biotechnol. 2: 37; Sassenfeld, H. M. (1990) Trends Biotechnol. 8: 88). In essence these modifications consist in fusing to the protein a polypeptide which has specific properties of interaction with a particular chromatographic support and which therefore gives the fusion protein the ability to be purified by affinity chromatography. One of these modifications consists in fusing to the protein in question a polyhistidine sequence which gives the fusion protein the ability to be purified by affinity chromatography using a support which contains divalent metal ions (Arnols, F. H. (1991) Bio/Technology 9: 151; Hochuli et al. (1988) Bio/Technology 6: 1321).
Although the obtaining of fusion proteins to improve the purification properties is in principle a simple and effective technique, its major disadvantage resides in the fact that the modification of a protein in its primary structure involves changes which may have an important effect on its secondary, tertiary and quaternary structure. These changes in structure may affect the protein to such an extent that it entirely or partially loses its functionality, converting it into a protein which is useless for the function that had been envisaged. The obtaining of a fusion protein therefore always entails great uncertainty, as the final result is largely unpredictable, especially when the first fusion experiment is being carried out, i.e. when the result of previous fusions is not known. It is in this uncertainty of the final result that the novelty of the process for obtaining a fusion protein lies, as it is not possible at present to predict with certainty what will happen when a protein fusion experiment is carried out.
In the scientific literature there is no description of any process for producing the DAO enzyme of T. variabilis genetically modified in such a way as to allow its purification in a single step and that it can be hyperproduced in an active form either in E. coli or in another microorganism.
For the description of this invention the starting point is the yeast T. variabilis ATCC 20931 as donor of deoxyribonucleic acid (hereinafter referred to as DNA). Once the genomic DNA of the yeast (which contains the gene with the genetic information relating to the production of DAO, hereinafter also called dao gene) had been obtained, it was used to construct a DNA library in E. coli using the phage vector xcex-GEM12. The analysis of the DNA library was performed by standard hybridization techniques using as probes synthetic oligonucleotides designed on the basis of regions of similarity found between different DAOs. In this way a series of recombinant clones of E. coli were isolated which contained a T. variabilis DNA fragment coding for the dao gene. The DNA fragment so obtained was subcloned in a plasmid vector obtained from a strain of E. coli. The recombinant vector was used to obtain the sequence of the DNA fragment which contained the dao gene of T. variabilis (SEQ ID NO: 1). Analysis of said sequence allowed characterization of the dao gene, which is structured in two exons and one intron.
As the DNA fragment previously obtained which contains the genomic sequence of the dao gene of T. variabilis has one intron, it cannot be used directly for its expression in E. coli. Steps were therefore taken to obtain a dao gene lacking said intron. For this purpose the dao gene was amplified by PCR using two synthetic oligonucleotides. The first of these was designed in such a way as to contain the following elements: a ribosome binding site, a translation initiation site, the complete sequence of the first exon and the first nucleotides of the 5xe2x80x2 end of the second exon. The second oligonucleotide contained the complementary sequence of the 3xe2x80x2 end corresponding to the second exon of the dao gene, including a translation termination codon. Different restriction sites useful for the cloning of DNA fragments were also included in these synthetic oligonucleotides. In this way a new DNA fragment was obtained which, after being isolated, was cloned in an E. coli plasmid vector (FIG. 1). Using the previously created restriction targets, the DNA fragment which contained the complete dao gene without the intron was subsequently subcloned in different E. coli plasmid vectors which had promoters that allowed the overexpression of genes in this host bacterium. The DAO activity produced by the various clones was assayed by colorimetric techniques and by HPLC chromatography. In this way recombinant clones of E. coli were obtained which produced a large amount of active DAO enzyme.
The dao gene was then modified by adding a nucleotide sequence coding for a polyhistidine. For this purpose a plasmid containing the dao gene was digested with a restriction enzyme which cut in the translation initiation codon, and the DNA ends resulting from the digestion were blunted with the Klenow fragment of the DNA polymerase I of E. coli. They were then ligated in the presence of a synthetic linker which contained the codons of the polyhistidine, with which a plasmid was produced that contained the modified dao gene in the 5xe2x80x2 end of its coding region (SEQ ID NO: 2). The recombinant plasmid so produced was transformed in a strain of E. coli and selected by hybridization techniques using the oligonucleotides of the polyhistidine linker as probes. The modified dao gene was sequenced in order to check that the desired fusion had been correctly produced.
The production of DAO modified with polyhistidine (hereinafter called hisDAO) was then studied, using various strains of E. coli as hosts of the previously constructed plasmids. In this way it was checked that the enzyme hisDAO was active.
For the production of hisDAO using the previously selected recombinant clones of E. coli, they are cultured in a medium containing a carbon source, a nitrogen source and mineral salts. The incubation temperature is between 18xc2x0 C. and 37xc2x0 C. and the pH must be maintained between 5 and 9. Flasks of various volumes, from 50 ml to 1000 ml, can be used for small-scale fermentation, with a quantity of medium between 10% and 50% of the volume of the flask. The duration of the fermentation can range from 12 to 90 hours.
The production of DAO by the recombinant microorganism can be improved if cultural conditions suitable for maintaining the stability of the recombinant vectors are chosen, which is achieved by adding to the culture medium the antibiotics for which the recombinant vector containing the dao gene shows a resistance marker (ampicillin, chloramphenicol, kanamycin, tetracycline, etc.). Apart from stabilizing the production, this prevents contamination of the culture medium by other undesirable microorganisms and also eliminates the strains which, because they have lost the recombinant vector, have stopped producing DAO.
The recombinant hisDAO-producing cells were separated from the culture medium by centrifugation and were then disrupted or permeabilized by means of chemical, enzymatic or mechanical processes. In order to obtain an enzymatic extract of greater purity the hisDAO was purified in a single step by affinity chromatography in columns of Co2+-IDA. For this purpose the crude enzymatic extract was loaded in a Co2+-IDA resin and the contaminant proteins were eluted by a washing with 20 mM phosphate buffer, pH 7.0, containing 0.2 M NaCl. The hisDAO enzyme was then eluted by a washing with 10 mM imidazole.
If Cu2+-IDA or Zn2+-IDA is used as the chromatographic support instead of Co2+-IDA, the hisDAO enzyme remains strongly bound to the matrix in active form without it being possible to elute it with high concentrations of imidazole. In this way the support, suitably washed to eliminate the contaminant proteins, can be used as an immobilized enzyme system.
Using the hisDAO enzyme purified from the recombinant clones of E. coli that expressed the chimeric gene, GL-7ACA was obtained from cephalosporin C.
The dialysed and concentrated enzymatic extracts obtained from the chromatography on the Co2+-IDA support can also be immobilized by making them react with other suitable inert solid supports, with the possibility of using immobilized hisDAO cyclically.
The novelty of this invention resides in the fact that this is the first time that the enzyme DAO of the yeast T. variabilis, modified as hisDAO, can be expressed in an active form in a prokaryotic microorganism such as E. coli and purified in a single step by affinity chromatography. In addition, an increase in the production of DAO relative to the quantity obtained in T. variabilis has been achieved, which facilitates the use of this enzyme on an industrial scale. The possibility of producing hisDAO in different strains of E. coli which do not have undesirable enzymes such as catalase or esterases, and also the possibility of improving the stability and catalytic properties thereof by means of further modifications by protein engineering techniques, are other aspects which increase the novelty concept of this patent.
The present invention, without any limitation, will be illustrated in greater detail in the Examples that are described below.